Time-to-response toxicity analysis as a method for drug susceptibility assessment in salmon lice

Abstract

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite causing infections of wild and farmed Atlantic salmon (Salmo salar L.) in the Northern hemisphere. While L. salmonis control at commercial mariculture sites increasingly employs non-medicinal approaches, such as cage designs reducing infection rates and biological control through cleaner fish, anti-parasitic drugs are still a requirement for effective fish health care. With only a limited range of salmon delousing agents available, all of which have been in use for more than a decade, drug resistance formation has been reported for different products. Successful resistance management requires reliable susceptibility assessment, which is usually achieved through L. salmonis bioassays. These tests involve the exposure of parasites to different drug concentrations and require significant numbers of suitable L. salmonis stages. The present study reports an alternative bioassay that is based on time-to-response toxicity analyses and can be carried out with limited parasite numbers. The assay determines the median effective time (ET50), i.e., the time required until impaired swimming and/or attachment behaviour becomes apparent in 50% of parasites, by conducting repeated examinations of test animals starting at the time point where exposure to a set drug concentration commences. This experimental approach further allows the estimation of the apparent drug susceptibility of individual L. salmonis by determining their time to response, which may prove useful in experiments designed to elucidate associations between genetic factors and the drug susceptibility phenotype of parasites. Three laboratory strains of L. salmonis differing in susceptibility to emamectin benzoate were characterised using standard 24 h bioassays and time-to-response toxicity assays. While both the median effective concentration (EC50) and the ET50 showed variability between experimental repeats, both types of bioassay consistently discriminated susceptible and drug-resistant L. salmonis laboratory strains.Statement of relevanceInfections by sea lice cause significant costs to the global salmon farming industry, which have been estimated to exceed €300 million per year worldwide. Control of sea lice still relies to a significant extent on chemical delousing; however, chemical control is threatened by resistance formation. Resistance can be combated by rotation between different drugs and strategic implementation of non-medicinal strategies. However, resistance management requires reliable and feasible methods of susceptibility assessment.The present study is a technical note introducing a novel approach to susceptibility assessments in sea lice. The method can be applied in susceptibility assessments on farms, where it offers the advantage of a reduced requirement of parasites for testing. In addition, the novel method allows deriving the times of parasite require to show a response after drug treatment has started, thus providing a variable characterizing the drug susceptibi...

Mutations in voltage‐gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis)

“…The laboratory‐maintained strains of L. salmonis used in this study were established from egg strings collected from Scottish salmon production sites . No drug selection has been applied during the isolation or maintenance of the strains.…”

“…Strain IoA‐00 was established in 2003 from an isolate originating in the Firth of Clyde and is susceptible to all current delousing agents including deltamethrin. Strain IoA‐01, derived in 2008 from material collected in Sutherland, is susceptible to deltamethrin but resistant to emamectin benzoate . Strains IoA‐02 and IoA‐03, which were established in 2011 and 2014, respectively, from the Shetland Islands and Sutherland, are resistant to emamectin benzoate and deltamethrin .…”

“…Strain IoA‐01, derived in 2008 from material collected in Sutherland, is susceptible to deltamethrin but resistant to emamectin benzoate . Strains IoA‐02 and IoA‐03, which were established in 2011 and 2014, respectively, from the Shetland Islands and Sutherland, are resistant to emamectin benzoate and deltamethrin . In bioassays involving 30 min of pesticide exposure and 24 h of recovery, the following deltamethrin median effective concentrations (EC 50 s), followed by 95% confidence limits, were determined in a previous study: IoA‐00: 0.28 µg L −1 (0.23–0.36 µg L −1 ), IoA‐01: 0.36 µg L −1 (0.26–0.46 µg L −1 ), IoA‐02: 40.1 µg L −1 (22.1–158.9 µg L −1 ), IoA‐03: >2.0 µg L −1 .…”

Genome-wide survey of cytochrome P450 genes in the salmon louse Lepeophtheirus salmonis (Krøyer, 1837)

“…Laboratory-cultured strains of L. salmonis investigated in this study have previously been described [19, 47]. Strain IoA-00 is susceptible to all current salmon delousing agents, whereas strain IoA-02 has previously been shown to be resistant against EMB and DM.…”

“…Azamethiphos susceptibility of the strains was determined in this study (see below). Since isolation, the strains have been cultured under identical conditions using Atlantic salmon ( Salmon salar L.) as host, as described in detail previously [17, 47]. All experimental infections were conducted under UK Home Office licence, and were subject to prior ethical review and appropriate veterinary supervision.…”